Liver disease affects millions of people worldwide. Fulminant hepatic failure is the clinical term for an immediate and catastrophic cessation in liver function, usually leading to death within a matter of hours. Other forms of liver disease, such as chronic hepatitis and cirrhosis, involve an insidious and progressive failure of liver function, with grim effects on physiological well-being and long-term prognosis. In the United States, there are an estimated 300,000 hospitalizations each year for liver disease, and 30,000 deaths—with only about 4,500 donor livers available for transplant.
A healthy liver has a remarkable ability to regenerate itself—but when this ability is compromised, the consequences are dire. An important challenge of modern medicine is to find a way to supplement the natural process of regeneration, and thereby restore liver function to affected patients.
Some early work has been done to identify liver progenitor cells in small animal models. Agelli et al. (Histochem. J. 29:205, 1997), Brill et al. (Dig. Dis. Sci. 44:364, 1999 and), and Reid et al. (U.S. Pat. No. 5,576,207) have proposed expansion conditions for early hepatic progenitor cells from embryonal and neonatal rat livers. Michalopoulos et al. (Hepatology 29:90, 1999) report a system for culturing rat hepatocytes and nonparenchymal cells in biological matrices. Block et al. (J. Cell Biol. 132:1133, 1996) developed conditions for expansion, clonal growth, and specific differentiation in primary cultures of hepatocytes induced by a combination of growth factors in a chemically defined medium. It has been known for some time that mature rat liver cells derive from precursors (sometimes referred to as “hepatoblasts” or “oval cells”) that have the capacity to differentiate into either mature hepatocytes or biliary epithelial cells (L. E. Rogler, Am. J. Pathol. 150:591, 1997; M. Alison, Current Opin. Cell Biol. 10:710, 1998; Lazaro et al., Cancer Res. 58:514, 1998; Germain et al., Cancer Res. 48:4909, 1988).
Unfortunately, a ready source of human hepatocytes for reconstitution therapy has not been identified. European Patent Application EP 953 633 A1 proposes a cell culturing method and medium for producing proliferated and differentiated human liver cells, apparently from donated human liver tissue. In most people's hands, the replication capacity of human hepatocytes in culture has been disappointing. As a remedy, it has been proposed that hepatocytes be immortalized by transfecting with large T antigen of the SV40 virus (U.S. Pat. No. 5,869,243).
A number of recent discoveries have raised expectations that stem cells may become a source of a variety of cell types and tissues for replacing those damaged in the course of disease, infection, or from congenital abnormalities. Various types of putative stem cells differentiate as they divide, maturing into cells that can carry out the unique functions of particular tissues, such as the heart, the liver, or the brain.
A particularly important development has been the isolation of two types of human pluripotent stem (hPS) cells from embryonic tissue. Pluripotent cells are believed to have the capacity to differentiate into most cell types in the body (R. A. Pedersen, Scientif. Am. 280(4):68, 1999). Early work on embryonic stem cells was done in mice (reviewed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 6:543, 1994). However, monkey and human pluripotent cells have proven to be much more fragile, and do not respond to the same culture conditions as mouse embryonic cells. It is only recently that discoveries were made that allow primate embryonic cells to be obtained and cultured ex vivo.
Thomson et al. (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully culture embryonic stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and International Patent Application WO 98/43679). Both hES and hEG cells have the long-sought characteristics of human pluripotent stem (hPS) cells: they are capable of ongoing proliferation in vitro without differentiating, they retain a normal karyotype, and they retain the capacity to differentiate to produce all adult cell types.
Spontaneous differentiation of pluripotent stem cells in culture or in teratomas generates cell populations with a heterogeneous mixture of phenotypes, representing a spectrum of different cell lineages. In a number of applications, it is desirable for differentiated cells to be of a more homogeneous nature—both in terms of the phenotypes they express, and in terms of the types of progeny they can generate.
Accordingly, there is a need for technology to generate more homogeneous differentiated cell populations from pluripotent embryonic cells of primate origin, particularly those from humans.